1. Field of the Invention
This invention relates to an assay involving genetically marked production host cell strains that are routinely the source of the manufacture of recombinantly produced proteins in a multiple-product fermentor facility. This invention also relates to the use of mixed cultures in a fermentor.
2. Description of Related Art
The potential for genetic markers in yeast strain identification has been recognized, and deliberately marked enological strains were developed by Vezinhet and Lacroix, Bull, O.I.V., 643-644, 759-773 (1984). Petering et al., Am. J. Enol. Vitic., 42: 6 (1991) discloses a procedure which utilizes recombinant DNA technology to introduce the E. coli .beta.-glucuronidase (GUS) gene as a marker into any desired yeast strain. Manufacture of new strains of microorganisms, which is applicable to microorganisms in which reproduction is normally asexual, is described in U.S. Pat. No. 2,820,742. The role of luciferase, along with its uses in luminescent assays and as a marker gene were reviewed by Ugarova et al., Biokhimiya, 58: 1351-1372 (1993). The luciferase gene can be inserted into contaminating bacteria using bacteriophages, and luciferase genes can be used as a marker in recombinant DNA studies, to assay promoter, and for other activities. Prosser et al., Critical Reviews in Biotechnology, 16: 157-183 (1996) discloses the development of techniques for detecting and tracking microorganisms in natural environments, and the development of molecular marker systems for such studies. In enology, the problem is to discriminate one strain from a huge variety of potential contaminants. Two approaches can be used: the use of an acquired characteristic, and genetic implantation of a discriminatory and easily localized property. The first is poorly suitable to vinification. The genetic labeling described in the above paper consists in the acquisition of antibiotic resistance (chloramphenicol and oligomycin) by the strain. Genetic determinism of this resistance is located in the cell mitochondrial genome. The labeling has the following characteristics: the nuclear genome is not modified; thus the risk of modifying the enologic potential of the strain is minimal, the acquired characteristics are easily recognized (growth on specific media), and no selective disadvantage is in natural competition for the labeled strain.
The advent of biotechnology has led to the clinical use and subsequent approval in the United States alone of more than ten recombinantly produced proteins, including gamma-interferon, beta-interferon, alpha-interferon, insulin, Factor VIII, tissue plasminogen activator, human growth hormone, colony stimulating factors, erythropoietin, and DNase. In addition, there are many drugs being developed or in the clinic for future approval. Capacity is limited in fermentation facilities, so that the same fermentor must be used for making different products. One-example is making different antibodies in the same fermentor. Similarly, an adjacent fermentor may simultaneously be producing a different product. For measures used to prevent the cross-contamination of licensed biological products manufactured in a multiuse facility and regulatory issues involved when facilities are used to manufacture more than one product, see Bader et al., "Multiuse Manufacturing Facilities for Biologicals," BioPharm, 5: 32-40 (September 1992).
There is a clear need for being able to detect whenever organisms producing a different product have contaminated the desired production culture. In a multiple-product facility, the contaminant could be any of several very similar organisms. The test must be simple to conduct and must sensitively detect any of the potential contaminants.